Sustained-release prostacyclin analog ONO-1301 ameliorates tubulointerstitial alterations in a mouse obstructive nephropathy model

Tubulointerstitial injuries are crucial histological alterations that predict the deterioration of renal function in chronic kidney disease. ONO-1301, a novel sustained-release prostacyclin analog, accompanied by thromboxane synthase activity, exerts therapeutic effects on experimental pulmonary hypertension, lung fibrosis, cardiomyopathy, and myocardial ischemia, partly associated with the induction of hepatocyte growth factor (HGF). In the present study, we examined the therapeutic efficacies of ONO-1301 on tubulointerstitial alterations induced by unilateral ureteral obstruction (UUO). After inducing unilateral ureteral obstruction in C57/BL6J mice, a single injection of sustained-release ONO-1301 polymerized with poly (D,L-lactic-co-glycolic acid) sustained-release ONO-1301 (SR-ONO) significantly suppressed interstitial fibrosis, accumulation of types I and III collagen, increase in the number of interstitial fibroblast-specific protein-1 (FSP-1)(+) cells, and interstitial infiltration of monocytes/macrophages (F4/80(+)) in the obstructed kidneys (OBK; day 7). Treatment with SR-ONO significantly suppressed the increase of the renal levels of profibrotic factor TGF-β and phosphorylation of Smad2/3, and elevated the renal levels of HGF in the OBK. In cultured mouse proximal tubular epithelial cells (mProx24), ONO-1301 significantly ameliorated the expression of fibroblast-specific protein-1 and α-smooth muscle actin as well as phosphorylation of Smad3 and increased the expression of zonula occludens-1 and E-cadherin in the presence of TGF-β1 as detected by immunoblot and immunocytochemistry, partly dependent on PGI(2) receptor-mediated signaling. Administration of rabbit anti-HGF antibodies, but not the control IgG, partly reversed the suppressive effects of SR-ONO on tubulointerstitial injuries in the OBK. Taken together, our findings suggest the potential therapeutic efficacies of ONO-1301 in suppressing tubulointerstitial alterations partly mediated via inducing HGF, an antifibrotic factor counteracting TGF-β.     

Protective effect of L-theanine on carbon tetrachloride-induced acute liver injury in mice

We studied effects of L-theanine, a unique amino acid in tea, on carbon tetrachloride (CCl(4))-induced liver injury in mice. The mice were pre-treated orally with L-theanine (50, 100 or 200 mg/kg) once daily for seven days before CCl(4) (10 ml/kg of 0.2% CCl(4) solution in olive oil) injection. L-theanine dose-dependently suppressed the increase of serum activity of ALT and AST and bilirubin level as well as liver histopathological changes induced by CCl(4) in mice. L-theanine significantly prevented CCl(4)-induced production of lipid peroxidation and decrease of hepatic GSH content and antioxidant enzymes activities. Our further studies demonstrated that L-theanine inhibited metabolic activation of CCl(4) through down-regulating cytochrome P450 2E1 (CYP2E1). As a consequence, L-theanine inhibited oxidative stress-mediated inflammatory response which included the increase of TNF-α and IL-1β in sera, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in livers. CCl(4)-induced activation of apoptotic related proteins including caspase-3 and PARP in mouse livers was also prevented by L-theanine treatment. In summary, L-theanine protects mice against CCl(4)-induced acute liver injury through inhibiting metabolic activation of CCl(4) and preventing CCl(4)-induced reduction of anti-oxidant capacity in mouse livers to relieve inflammatory response and hepatocyte apoptosis.     

The effects of thromboxane A2 inhibitors (OKY-046 and ONO-3708) and leukotriene inhibitors (AA-861 and LY-171883) on CCl4-induced chronic liver injury in mice

The effects of OKY-046, a selective thromboxane A2 (TxA2) synthetase inhibitor, ONO-3708, a novel TxA2 receptor antagonist, AA-861, a selective 5-lipoxygenase inhibitor and LY-171883, a peptide leukotrienes (p-LTs) receptor antagonist on the chronic liver injury were investigated in mice. The chronic liver injury was induced by the injection of carbon tetrachloride (CCl4) two times a week for twelve weeks in mice. In chronic liver injury models, significant histopathological changes in the liver and extensive elevation of glutamate transaminase (GOT and GPT) activity were observed. Administration of OKY-046, ONO-3708, AA-861 and LY-171883 for 12 weeks suppressed the elevation of serum GOT and GPT levels and histopathological changes in CCl4-induced chronic liver injury. These results suggest that TxA2 and LTs inhibitors are effective for the onset and development of chronic liver injury in mice.     

CCl4-induced acute liver injury in mice is inhibited by hepatocyte growth factor overexpression but stimulated by NK2 overexpression

Hepatocyte growth factor (HGF) inhibits acute liver injury. NK2 acts as an antagonist to HGF in vitro, but its in vivo function has reached no consensus conclusions. We have investigated in vivo effects of HGF and NK2 on CCl4-induced acute liver injury. Elevation of the serum alanine aminotransferase level and extension of centrilobular necrosis were inhibited in HGF transgenic mice but were promoted in NK2 transgenic mice. Hepatocyte proliferation after liver injury was not inhibited in NK2 transgenic mice. Thus, this study indicates that HGF inhibits liver injury, and NK2 antagonizes HGF on liver injury, however, NK2 may not antagonize HGF on hepatocyte proliferation.     

蟛蜞菊内酯对四氯化碳致小鼠肝损伤的保护作用

目的:研究中药活性物质蟛蜞菊内酯的保肝作用及其机制。方法:采用小鼠腹腔注射CCl4制作肝损伤模型,测定小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、丙二醛(MDA),谷胱甘肽(GSH)和超氧化物歧化酶(SOD)指标,进行肝脏的组织病理学检查,观察蟛蜞菊内酯对CCl4所致肝损伤的保护作用。结果:蟛蜞菊内酯能明显降低肝损伤小鼠的血清ALT、AST和肝组织匀浆中MDA含量,SOD活力增强,明显减轻肝组织变性。结论蟛蜞菊内酯对CCl4引起的肝损伤有明显的保护作用,其机制可能与其抗氧化作用有关。     

4-羟基苯并恶唑-2-酮对四氯化碳致小鼠急性肝损伤保护作用

日的研究4一羟基苯并恶唑-2-酮（4-hydroxy-2-benzoxazolone,HBOA）对四氯化碳所致小鼠急性肝损伤的保护作用,并探讨其疗效机制。方法采用腹腔注射四氯化碳（carbonte trachloride,cch）制备小鼠急性肝损伤模型,HBOA灌胃给药,检测小鼠血清中的乳酸脱氢酶（LDH）活性以及肝组织中过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）含量,并用免疫组化法观察肿瘤坏死因子（TNF-a）的表达情况。结果HBOA能明显降低CCh致急性肝损伤小鼠血清LDH活性,同时升高肝组织中CAT、GSH-Px的活性并降低肝组织中TNF-a的表达。结论HBOA对CCh所致小鼠急性肝损伤有一定的保护作用。     

Prostacyclin agonist with thromboxane synthase inhibitory activity (ONO-1301) attenuates bleomycin-induced pulmonary fibrosis in mice

The balance between prostacyclin and thromboxane A2 (TXA2) plays an important role in pulmonary homeostasis. However, little information is available regarding the therapeutic potency of these prostanoids for pulmonary fibrosis. We have recently developed ONO-1301, a novel long-acting prostacyclin agonist with thromboxane synthase inhibitory activity. Thus we investigated whether repeated administration of ONO-1301 attenuates bleomycin-induced pulmonary fibrosis in mice. After intratracheal injection of bleomycin or saline, mice were randomized to receive repeated subcutaneous administration of ONO-1301 or vehicle. Bronchoalveolar lavage (BAL) and histological analyses were performed at 3, 7, and 14 days after bleomycin injection. In vitro studies using mouse lung fibroblasts were also performed. ONO-1301 significantly attenuated the development of bleomycin-induced pulmonary fibrosis, as indicated by significant decreases in Ashcroft score and lung hydroxyproline content. ONO-1301 significantly reduced total cell count, neutrophil count, and total protein level in BAL fluid in association with a marked reduction of TXB2. A single administration of ONO-1301 significantly increased plasma cAMP level for >2 h. In vitro, ONO-1301 and a cAMP analog dose-dependently reduced cell proliferation in mouse lung fibroblasts. The reduction in cell proliferation by ONO-1301 was attenuated by a protein kinase A (PKA) inhibitor. Furthermore, bleomycin mice treated with ONO-1301 had a significantly higher survival rate than those given vehicle. These results suggest that repeated administration of ONO-1301 attenuates the development of bleomycin-induced pulmonary fibrosis and improves survival in bleomycin mice, at least in part by inhibition of TXA2 synthesis and activation of the cAMP/PKA pathway.     

聚乙二醇修饰重组细胞珠蛋白对小鼠急性肝损伤的保护作用

旨在探究聚乙二醇修饰重组细胞珠蛋白(PEG modified recombinant cytoglobin,PEG-rCygb)对小鼠急性肝损伤的保护作用。采用CCl4诱导KM小鼠急性肝损伤模型,尾静脉注射PEG-rCygb,收集血清及肝脏组织检测各项生化指标及组织病理学变化。结果表明,PEG-rCygb治疗组小鼠肝脏系数减小,血清中AST﹑ALT水平降低,肝组织匀浆中MDA含量减少,GSH含量增加,T-SOD、CAT活性升高。肝组织切片HE染色显示PEG-rCygb可以缓解肝细胞脂肪变性,减少炎症因子,减轻肝细胞损伤。体外细胞学实验表明rCygb经PEG修饰后对H2O2造成的肝星状细胞(HSC)氧化损伤发挥的保护作用增强。研究结果显示PEG-rCygb提高了机体对自由基的清除能力,对CCl4引起的小鼠急性肝损伤具有保护作用。     

肝细胞生长因子对四氯化碳损伤肝细胞的保护作用及相关基因表达

利用无血清原代培养大鼠肝细胞,观察重组人肝细胞生长因子（ｒｈＨＧＦ）对ＣＣｌ４染毒肝细胞的保护作用。结果表明：（１）ｒｈＨＧＦ（５ｎｇ／ｍｌ）预自理后可显著提高ＣＣｌ４（１５ｍｍｏｌ／Ｌ）染毒肝细胞存活率,降低细胞内丙氨酸氨基转移酶（ＡＬＴ）、Ｋ＾＋的漏出;（２）表皮生长因子（ＥＧＦ,５０ｎｇ／ｍｌ）和ｒｈＨＧＦ（５ｎｇ／ｍｌ）合用预处理肝细胞,ＣＣｌ４染毒后细胞内ＡＬＴ、Ｋ＾＋漏出较ｒｈＨＧＦ和     

Role of nitric oxide in the regulation of fibrogenic factors in experimental liver fibrosis in mice

Previously, we have shown that an increased expression level of iNOS but a reduction in the expression of eNOS is associated with increased oxidative stress markers in CCl?-induced experimental liver fibrosis. The present study aimed to investigate the effect of L-arginine and 5-methylisothiourea hemisulfate (SMT) in the expression of profibrogenic factors in chronic liver injury. ICR mice were treated with CCl? with or without treatment of L-arginine, an NO donor, or SMT, an iNOS inhibitor. The expression of matrix metalloptroteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR. The activity of the MMP-2 and MMP-9 were measured by zymography. Our results showed that CCl?-treated mice showed significant up-regulation of expression of pro-fibrogenic factors, TNF-α and COX-2. Treatment with L-arginine or SMT showed a significant reduction in CCl?-induced expression of these pro-fibrogenic factors, TNF-α and COX-2. In conclusion, both SMT and L-arginine effectively attenuated the progression of CCl?-induced liver fibrosis. SMT suppresses iNOS mediated NO production. However, L-arginine augments NO production. The similar effect of the two drugs on liver fibrosis indicates that there may be two distinct pathways of NOS mediated fibrogenesis in chronic liver injury by iNOS and eNOS. Our results suggest that eNOS-mediated liver fibrogenesis may play a more important role than that of iNOS in chronic liver injury. Taken together, these results support the contention that NO plays an active role in the progression of liver fibrosis and hepatocellular damage.     

铁棍山药多糖对四氯化碳诱导的急性小鼠肝损伤的保护作用

为了研究铁棍山药(D.oppositacv.Tiegun)多糖对四氯化碳诱导的小鼠急性肝损伤的保护作用,取72只昆明小鼠随机分为对照组,四氯化碳(CCl4)模型组,阳性对照(联苯双酯)组,铁棍山药多糖低、中、高剂量组,每组12只,灌胃处理后使用CCl4制备急性肝损伤小鼠模型,观察各组形态学变化,同时测定生化指标。实验结果显示,经铁棍山药多糖处理的小鼠的肝损伤程度明显轻于模型组,铁棍山药多糖能降低小鼠血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)含量,提高超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性,降低丙二醛(MDA)、一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)的含量。本研究结果表明,铁棍山药多糖对CCl4所诱导的小鼠肝损伤起到一定的保护作用。     

Sustained-Release Delivery of Prostacyclin Analogue Enhances Bone Marrow-Cell Recruitment and Yields Functional Benefits for Acute Myocardial Infarction in Mice

Background

A prostacyclin analogue, ONO-1301, is reported to upregulate beneficial proteins, including stromal cell derived factor-1 (SDF-1). We hypothesized that the sustained-release delivery of ONO-1301 would enhance SDF-1 expression in the acute myocardial infarction (MI) heart and induce bone marrow cells (BMCs) to home to the myocardium, leading to improved cardiac function in mice.

Methods and Results

ONO-1301 significantly upregulated SDF-1 secretion by fibroblasts. BMC migration was greater to ONO-1301-stimulated than unstimulated conditioned medium. This increase was diminished by treating the BMCs with a CXCR4-neutralizing antibody or CXCR4 antagonist (AMD3100). Atelocollagen sheets containing a sustained-release form of ONO-1301 (n = 33) or ONO-1301-free vehicle (n = 48) were implanted on the left ventricular (LV) anterior wall immediately after permanent left-anterior descending artery occlusion in C57BL6/N mice (male, 8-weeks-old). The SDF-1 expression in the infarct border zone was significantly elevated for 1 month in the ONO-1301-treated group. BMC accumulation in the infarcted hearts, detected by in vivo imaging after intravenous injection of labeled BMCs, was enhanced in the ONO-1301-treated hearts. This increase was inhibited by AMD3100. The accumulated BMCs differentiated into capillary structures. The survival rates and cardiac function were significantly improved in the ONO-1301-treated group (fractional area change 23±1%; n = 22) compared to the vehicle group (19±1%; n = 20; P = 0.004). LV anterior wall thinning, expansion of infarction, and fibrosis were lower in the ONO-1301-treated group.

Conclusions

Sustained-release delivery of ONO-1301 promoted BMC recruitment to the acute MI heart via SDF-1/CXCR4 signaling and restored cardiac performance, suggesting a novel mechanism for ONO-1301-mediated acute-MI heart repair.     

Transgene expression of human PON1 Q in mice protected the liver against CCl4-induced injury

BACKGROUND: Oxidative stress, often in association with decreased antioxidant defenses, plays a pathogenetic role in both initiation and progression of liver injuries, leading to almost all clinical and experimental conditions of chronic liver diseases. Human paraoxonase 1 (hPON1) is a liver-synthesized enzyme possessing antioxidant properties. Here, we investigate the effects of transgene-expressed hPON1 Q on alleviating lipid peroxidation and preventing liver injury in a mouse model. METHODS: The hPON1 Q gene was cloned into pcDNA3.0 plasmid and electro-transferred into mouse skeletal muscle. After CCl4 had been administrated to induce liver injury, mice were monitored for serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malonyldialdehyde (MDA). The extent of CCl4-induced liver injury was also analyzed through histopathological observations. RESULTS: After gene delivery, hPON1 mRNA expression was detected in mouse muscle and serum PON1 activity was 1.5 times higher than that of the control counterpart. In the PON1 Q gene transferred mice, protection against CCl4-induced liver injury was reflected by significantly decreased serum ALT, AST and MDA levels compared to those in control mice (P < 0.01). Histological observations also revealed that hepatocyte necrosis, hemorrhage, vacuolar change and hydropic degeneration were apparent in control mice after CCl4 administration. In contrast, the damage was significantly prevented (P < 0.01) in the hPON1 Q transferred mice. CONCLUSIONS: Intramuscular electro-transfer of the hPON1 Q gene led to efficient expression of hPON1 in mice. Elevated levels of PON1, by virtue of its potency to alleviate oxidative stress, could protect mice from suffering CCl4-induced liver damage.     

冬虫夏草菌丝提取物对化学性肝损伤的辅助保护作用

为明确冬虫夏草菌丝提取物对急性肝损伤小鼠谷丙转氨酶（ALT）、谷草转氨酶（AST）、肝细胞变性及坏死程度的影响,采用四氯化碳（CCl4）诱导小鼠急性化学性肝损伤模型,将动物随机分成5组,分别是空白对照组、模型组、冬虫夏草菌丝提取物低剂量组（1.11g/kg BW）、中剂量组（3.33g/kg BW）、高剂量组（10.00g/kg BW）,检测血清ALT、AST值,并取肝脏作病理切片,观察肝脏的病理损伤情况。冬虫夏草菌丝提取物高剂量组能明显降低CCl4急性肝损伤小鼠血清ALT值,减轻肝细胞坏死程度,表明冬虫夏草菌丝提取物对化学性肝损伤有辅助保护功能。     

The role of thromboxane A2 [TxA2] in liver injury in mice

The role of thromboxane A2 (TxA2) in CCl4-induced liver disease was investigated in mice. Significant elevation of TxB2 in the liver was observed 6 hours after the injection of CCl4. Administration of OKY-046, a selective TxA2 synthetase inhibitor (10 and 50 mg/kg) and ONO-3708, a TxA2 receptor antagonist, (0.5, 1 and 2 mg/Kg) suppressed the elevation of serum GOT and GPT levels and histopathological changes of the liver. In addition, OKY-046 inhibited the elevation of TxB2 in the liver. When U-46619, a stable TxA2 mimetic was injected i.v. into the mice, clear elevation of serum GOT and GPT levels and histopathological score of the liver were observed. These results suggest that TxA2 play a role for the onset of CCl4-induced liver injury in mice.     

Intermittent administration of a sustained-release prostacyclin analog ONO-1301 ameliorates renal alterations in a rat type 1 diabetes model

Diabetic nephropathy is the most common pathological disorder predisposing end-stage renal disease. ONO-1301 is a novel sustained-release prostacyclin analog possessing thromboxane (TX) synthase inhibitory activity. Here, we aimed to investigate the therapeutic efficacies of ONO-1301 in a rat type 1 diabetic nephropathy model. Streptozotocin (STZ)-induced diabetic rats received injections of slow-release form of ONO-1301 (SR-ONO) every 3 weeks. Animals were sacrificed at Week 14. SR-ONO significantly suppressed albuminuria, glomerular hypertrophy, mesangial matrix accumulation, glomerular accumulation of monocyte/macrophage, increase in glomerular levels of pro-fibrotic factor transforming growth factor (TGF)-beta1 and the number of glomerular alpha-smooth muscle actin (SMA)(+) cells in diabetic animals. The glomerular levels of hepatocyte growth factor (HGF) were significantly increased in SR-ONO-treated diabetic animals. Taken together, these results suggest the potential therapeutic efficacy of intermittent administration of SR-ONO in treating diabetic nephropathy potentially via inducing HGF, thus counteracting the pro-fibrotic effects of TGF-beta1.     

Hepatoprotective effects of berberine on liver fibrosis via activation of AMP-activated protein kinase

Aims

We investigated the protective effect of berberine (BBR) on chronic liver fibrosis in mice and the potential mechanism underlying the activation of AMP-activated protein kinase (AMPK) pathway.

Main methods

CCl4-induced chronic liver fibrosis model in mice was established and activated rat hepatic stellate cell was treated with BBR. Cell viability was evaluated by SRB assay and protein expressions were detected by Western blot.

Key findings

Our results showed that BBR ameliorated the liver fibrosis in mice with CCl4-induced liver injury and inhibited the proliferation of hepatic stellate cell in dose- and time-dependent manner. BBR decreased the enzyme release of ALT, AST, and ALP in serum, elevated SOD and reduced MDA content of liver tissue in CCl4-induced liver fibrosis model. BBR delayed the formation of regenerative nodules and reduced necrotic areas compared to CCl4 group. Moreover, BBR treatment activated AMPK, decreased the protein expression of Nox4, TGF-β1 and the phosphorylated Akt. The expression of smooth muscle actin (α-SMA), the marker of activated hepatic stellate cell, was also reduced by BBR treatment.

Significance

Our studies firstly demonstrated that BBR exerted hepatoprotective effects possibly via activation of AMPK, blocking Nox4 and Akt expression. Our findings may benefit the development of new strategies in the prevention of chronic liver disease.     

Protective role of estrogen-induced miRNA-29 expression in carbon tetrachloride-induced mouse liver injury

Previous studies have indicated that female animals are more resistant to carbon tetrachloride (CCl(4))-induced liver fibrosis than male animals, and that estradiol (E(2)) treatment can inhibit CCl(4)-induced animal hepatic fibrosis. The underlying mechanism governing these phenomena, however, has not been fully elucidated. Here we reported the role of estrogen-induced miRNA-29 (miR-29) expression in CCl(4)-induced mouse liver injury. Hepatic miR-29 levels were differentially regulated in female and male mice during CCl(4) treatment. Specifically, the levels of miR-29a and miR-29b expression were significantly decreased in the livers of male, but not female, mice following 4 weeks of CCl(4) treatment. The down-regulation of miR-29a and miR-29b in male mouse livers correlated with the early development of liver fibrosis, as indicated by increased expressions of fibrotic markers in male mice relative to female mice. In addition, E(2) was maintained at a higher level in female mice than in male mice. In contrast to TGF-β1 that decreased miR-29a/b expression in murine hepatoma IAR20 cells and normal hepatocytes, E(2) enhanced the expression of miR-29a/b through suppression of the nuclear factor-κB (NF-κB) signal pathway, which negatively regulates miR-29 expression. Furthermore, both E(2) treatment and intravenous injection of the recombinant adenovirus expressing miR-29a/b markedly increased the miR-29a/b level and attenuated the expression of fibrotic markers in mouse livers during CCl(4) treatment, supporting the protective role of E(2)-induced miR-29 in CCl(4)-induced hepatic injury. In conclusion, our results collectively demonstrate that estrogen can inhibit CCl(4)-induced hepatic injury through the induction of hepatic miR-29.